The proposed research is designed to gain detailed information about the molecular structure of plasma membranes. It is generally accepted that biomembranes are composed primarily of proteins and amphiphilic lipids arranged in bimolecular arrays and that these bilayers will separate into two portions, "halves," upon freezing and fracturing. I have developed a "bulk" splitting method to produce membrane fractions enriched in outer or inner halves, am using it to analyze the transmembrane distribution of cholesterol in human erythrocytes (RBC's), and will continue to use it to study other RBC lipids and polypeptides. A new electron microscopic method that combines monolayer freeze-fracture (just described) with electron microscopic autoradiography (MONOFARG) is currently being developed to examine the in-plane location of radioisotopic molecules as well as the kinetics of their transmembrane diffusion. Finally, a third method, also based on non-random splitting of oriented planar membranes, that utilized double-labeled membrane splitting techniques is being developed. These three techniques can provide information about membrane association native molecules or synthetic probes not easily studied by other methods. When developed, the double-label approach can provide information about the transmembrane distribution of clinically important molecules with the rapidity appropriate to a diagnostic tool.